Gene transfer by nonviral methodologies (i.e. lipofection) has been found to be very inefficient in nondividing cell populations. Unfortunately, cells are not actively dividing in many in vivo tissues that are potential clinical targets for gene therapy. While receptor targeting, fusigenic peptides, or endosome disrupting agents help overcome some of the first barriers that limit liposome-based gene delivery, thus far virus free gene transfer using liposomes has had limited clinical utility because of the difficulty of transporting genetic material into the nucleus of a nondividing cell.
Cationic liposomes are commonly used as a means of delivering DNA to dividing cells in culture. DNA/lipid aggregates form spontaneously after mixing aliquots of the liposome reagent with an aqueous solution of DNA (Felgner et al. Proc. Natl. Acad. Sci. U.S.A. 1987 84, 7413; Felgner et al. J. Biol. Chem. 1994 269, 2550; Felgner, P. L. and Ringold, G. M. Nature 1989 337, 387). However, the efficiency of transfection is low with nondividing cells and certain target cells such as endothelium. For example, COS-7 cells lipofected with chloramphenicol acetyl transferase (CAT) gene are nearly 80% CAT positive compared to essentially no CAT expression in endothelial cells (Nathwani et al. Brit. J. Haem. 1994 88, 121).
Arterial gene transfer has been proposed for the treatment of atherosclerosis and restenosis following angioplasty. The two major strategies of arterial gene transfer are: (1) the cultivation, transfection and reintroduction of autologous endothelial cells to the recipient, (Dichek et al. Circulation 80, 1347, 1989; Wilson et al. Science. 244, 1344, 1989) and (2) the direct catheter delivery of genetic material with transfection vehicle or virus to the cells of the artery (Nabel et al. Science 244, 1342, 1989; Lemarchand et al. Proc. Natl. Acad. Sci. 89, 6482, 1992; Flugelman et al. Circulation. 85, 1110, 1992; Takeshita et al. J. Clin. Invest. 93, 652, 1994; Nabel E. G., J. Vasc. Sur., 15, 931, 1992; Chapman et al. Circ. Res. 71, 27, 1992; Schulick et al. Circ. Res. 77, 475, 1995). While receptor targeting of vectors to endothelium may provide benefit, the endothelium is ideally accessible by catheter. In vivo, endothelial cells have a very low mitotic rate. Hence, while adenoviral gene transfer (Flugelman et al. Circ. 1992 85:1110-1117; Nabel et al. Science 1989 244:1342-1344; Subramanian, A. and Diamond, S. L. Tissue Engineering 1997 3:39-52; Lemarchand et al. Circ. Res. 1993 72:1132-1138; Steg at al. Circ. 90:1648-1653; Sung et al. Circ. Res. 1993 73:797-807; Schachtner et al. Circ. Res. 1995 76: 701-709) is highly efficient in transfecting these cells, gene transfer via liposome mediated routes and retroviral vectors which are more efficient in mitotic cells (Flugelman et al. Circulation. 85, 1110, 1992 1992) result in low levels of gene transfer.
Thus, various attempts have been made to improve efficiency of gene transfer of nondividing cells via liposome mediated routes.
Recent improvements of lipofection protocols for subconfluent epithelium tend to plateau at about 30% transfection efficiency: α5β1 integrin targeting peptide results in 25% transfection of corneal endothelium (Hart et al. Hum. Gene Ther. 1998 9:575-585); replication-deficient AdV-complexed plasmid results in 20% transfection of HUVEC (Edgell et al. Biotechniques 1998 25:264-268) and 25 to 35% transfection of BAEC (Go et al. Am. J. Physiol. 1998 274:H1-H7); histone complexation of plasmid results in 20% transfection of bovine aortic endothelial cells (BAEC) (Subramanian, A. and Diamond, S. L. Tissue Engineering 1997 3:39-52); AdV fiber added to plasmid results in 30% transfection of BAEC (Hong et al. Chin. Med. J. 1995 108:332-338) and plasmid condensed with recombinant histone H1 containing SV40 T-antigen NLS resulted in 10 to 30% transfection of COS-7 or NIH3T3 (Fritz et al. Hum Gene Ther. 1996 7:1395-1404). It is not known whether the benefits of these protocols can be achieved when using confluent cells at the time of transfection. The 30% plateau is believed to represent the persistence and elevated level of intact cytoplasmic plasmid available to accomplish gene transfer in cells dividing at times one to two days after transfection. An unprotected plasmid with short half-life in the cytoplasm would transfect only the cells dividing in the first few hours after the lipofection.
A large amount of labeled-plasmid transfected into the cells via these protocols was found to be present in the endosomes as indicated by punctate staining. However when the plasmids were transcribed cytoplasmically using a T7 RNA polymerase based system, 80% of cells were found to express β-galactosidase, indicating that some of the plasmid does get out of the endosome into the cytoplasm in the cells.
However, plasmid DNA cannot readily enter the nucleus since they are typically excluded by the nuclear pore (Felgner et al. Proc. Natl. Acad. Sci. USA 1987 84:7413-7417; Felgner, P. L. and Ringold, G. M. Nature 1987 337:387-388; Jo et al. J. Biol. Chem. 1997 272:1395-1401; and Zabner et al. J. Biol. Chem. 1995 270:189997-19007). Increasing cytoplasmic concentrations of plasmid can directly enhance total expression in dividing cells by enhancing plasmid levels in the nucleus, post-mitotically. It is believed that the frequency of nuclear import events in nondividing cells increases with elevated plasmid levels through enhancement of the probability of plasmid encounter with the nuclear pore entrance. However, inefficient nuclear pore targeting due to cytoplasmic sequestration (scaffolding) and inefficient transit of plasmid across the pore remain important rate limits during nonviral gene transfer.
Attempts to conjugate classical nuclear localization signals (NLS) to a plasmid to alleviate this problem have had limited success (Subramanian, A. and Diamond, S. L. Tissue Engineering 1997 3:39-52; Fritz et al. Hum. Gene Ther. 1996 7:1395-1404). Classical nuclear localization signals involve peptide sequences of clustered residues that interact with two proteins, importin-α and importin-β, also known as karyopherin α and karyopherin β, respectively. The protein importin-β binds the nuclear pore. The importin complex also binds a GTPase RNA (Nakielny et al. Exp. Cell Res. 1996 229:261-266). Examples of classical NLS include SV40 large T antigen (PPKKKRKV; SEQ ID NO:6), adenovirus E1A (SCKRPRP; SEQ ID NO:7), human lamin A (SVTKKRKL; SEQ ID NO:8); polyoma large T antigen (PPKKARED; SEQ ID NO:9), polyoma large T antigen (VSRKRPRP; SEQ ID NO:10), human c-myc (PAAKRVKL; SEQ ID NO:11), rat glucocorticoid receptor (RKTKKKIK; SEQ ID NO:12), and human estrogen receptor (IRKDRRG; SEQ ID NO:13). The classical nuclear localization sequence can also contain a bipartite form, with two basic amino acids separated by an amino acid spacer from a second cluster of three or more basic amino acids. A prototypical bipartite NLS is found in nucleoplasmin (AVKRPAATKKAGQAKKKKLD; SEQ ID NO:14).
It has been found that histones (Subramanian, A. and Diamond, S. L. Tissue Engineering 1997 3:39-52; Fritz et al. Hum. Gene Ther. 1996 7:1395-1404) or SV40 T antigen (Sebestyen et al. Nature Biotech. 1998 16:80-85) when linked covalently or by charge interactions to plasmid have not been able to get plasmid across the nuclear pore of intact cells to effect fully efficient gene transfer with 100% transfection.
In the present invention methods and compositions are providing for delivering selected molecules to the nuclei of eukaryotic cells via nuclear targeting peptides containing nonclassical nuclear localization signals.